GSM7977988: scRNA lungs, PRDM3/16KO, Rep2; Mus musculus; RNA-Seq
Fetal lung tissue was stored on ice in HypoThermosol FRS preservative solution (StemCell Technologies). Two lungs of the same genotype were minced with a razor blade for 2min. 25mg of tissue was digested on ice for 4min in 1mL of Collagenase/Elastase/Dispase enzyme mix (6mg/mL Collagenase A (Sigma, 10103578001), 4.3 units/mL Elastase (Worthington, LS002292), 9 units/mL Dispase (Worthington, LS02100) 5mM CaCl2, 125 units/mL DNase (Applichem, A3778), in DPBS (ThermoFisher, 14190144). Tissue was placed in a sterile petri dish for a further 2min of mincing. The cell suspension was pipetted into an Eppendorf tube on ice and the petri dish was rinshed with an additional 500mL of enzyme mix. The mixutre was triturated and shaken for 2min. Large tissue chunks were allowed to settle and the top 1200uL of supernatnat passed through a 30um filter (Miltenyi 130-098-458) on a 15mL conical tube. The filter was rinsed with 5mL ice cold PBS with 0.04% BSA. Remaining tissue was further digested with 1mL enzyme mix containing 10mg/mL Bacillus Licheniformis (Sigma, P5380) and 0.5mM EDTA in DPBS with periodic trituration for an additonal 10min. Resulting cell suspension was passed through 30um filter with 7mL PBS/BSA for rinsing the filter. Cells were pelleted at 300xg for 5min at 4C and supernatate was aspirated. Cells were resuspended in 900uL Red Blood Cell Lysis Bufer (Sigma, R7757) and incubated on ice for 2min. 12mL PBS/BSA was added, then cells pelleted at 200xg for 5min at 4C. Supernatant was asipirated and cells resuspended in 1mL PBS/BSA. Cells were counted using a hemacytometer with trypan blue and concentration adjusted to 1000cell/mL prior to seperation using the Chromium 10X Platform. Cell separation and cDNA synthesis performed by Cincinnati Children's Hospital Single Cell Genomics Facility Libraries generated and sequenced by Cincinnati Children's Hosptial Genomics Sequencing Facility